Calorie restriction (CR) is a dietary regimen that extends the maximum life span of a wide variety of organisms. CR also protects against aging-associated diseases, including type II diabetes and cancer. There are several theories to explain how CR protects against the detrimental effects of aging, but the molecular mechanism has yet to be elucidated. Therefore, one of the major goals of aging research at this time is to understand how CR works, which will help identify future therapeutic targets for the prevention or treatment of age-related disease. Remarkably, there is a yeast model of CR that consists of simply reducing the glucose in the growth medium from 2% to 0.5% (or less), which leads to an extension of replicative life span (RLS), defined as the number of times that a mother cell can divide, and chronological life span (CLS), defined as the number of days that cells survive in a non-dividing state. SIR2 is a central player in the regulation of yeast RLS and calorie restriction, but is not a key regulator of chronological longevity or CR-mediated extension of CLS. Therefore, the long-term goal of this project is to use the yeast CLS system for the identification and molecular-genetic dissection of novel CR-mediated longevity pathways. To facilitate rapid analysis of multiple mutants and growth conditions, the CLS assay has been optimized such that -100 samples can be processed at the same time. The specific aims of the project first focus on the purification and identification of an extracellular longevity factor that modulates yeast life span. In addition, we propose utilizing a high throughput genetic screen to identify genes that function in calorie restriction-mediated extension of CLS. The last specific aim is focused on dissecting the role of the highly conserved de novo purine synthesis pathway in modulating chronological and replicative life span in response to CR. Given the large number of genes and basic cellular processes shared between yeast and human cells, this study is expected to generate data that is highly applicable to CR research in mammals.